The present invention relates to the use of hCG, hCG xcex2-subunit, as well as fragments and active derivatives thereof, or hCG xcex2-core in the preparation of a medicament for the prophylaxis and/or the treatment of pathologies requiring inhibition of the matrix metalloprotease (MMP) enzymatic activity.
Several pathological processes involve tissue remodeling and degradation, including tumor invasion, joint destruction in rheumatoid arthritis and osteoarthritis, periodontotitis and angiogenesis-dependent diseases such as corneal neovascularization, diabetic retinopathy, neovascular glaucoma, hemangioma, psoriasis, scleroderma and solid tumors.
Tumor invasion is a complex multistage process involving: a) tumor cell exit from the primary tumor and transit across host basement membrane; b) penetration (intravasation) and transit through the vascular system; c) arrest at distal site in the vasculature and exit from the circulation by repenetration of endothelial basement membrane (extravasation); d) entry into target tissue and proliferation at the secondary site.
One of the key events necessary to allow the passage of tumor cells is the removal of the extracellular matrix (ECM) by ECM degrading enzymes secreted by metastatic tumor cells and/or tumor-activated host stroma cells (D. J. Dodwell et al., 1993).
Basement membrane and interstitial connective tissue make up the ECM, which is composed by collagen, proteoglycans and adhesion glycoproteins. ECM provides a physical support to cells and tissues and cell-ECM interactions regulate cell growth, differentiation and migration.
ECM degrading enzymes include matrix metalloproteases (MMPs), urokinase, tissue plasminogen activator, cathepsins, trypsins and heparanases.
MMPs, which are secreted as proenzymes and require activation, comprise three subclasses, based on substrate preference: interstitial collagenases, gelatinases or type IV collagenases and stromelysins (reviewed by J. F. Woessner, 1991, see in particular Table 1).
A positive correlation between overexpression of MMPs (mostly type IV collagenases and stromelysin 3) and tumor aggressiveness has been established for several types of tumors (see I. C. Anderson et al, 1995; B. L. Lokeshwar et al, 1993; W. C. Powell et al, 1993; and J. M. Ray, 1995).
In rheumatoid arthritis and osteoarthritis, infiltrating monocytes/macrophages, inflammatory cytokine-activated synovial fibroblasts and chondrocytes produce abnormal amounts of collagenase which contributes to cartilage degradation and progression of the disease (A. M. M. Miltenburg et al, 1995).
MMPs are also involved in normal and pathological angiogenesis, as endothelial cells have to degrade the ECM to exit from the parent vessel and initiate a new capillary network (R. B. Vernon et al., 1995).
It has also been suggested that MMPs may play a role in multiple sclerosis in connection with blood-brain barrier injury (G. A. Rosenberg et al., 1996).
Taken together the above data suggest that MMPs may represent a therapeutical target in many diseases. Accordingly, various strategies based on natural or synthetic compounds are being developed to inhibit MMP activity.
ECM degradation is a tightly regulated process and collagenase proteolysis is controlled following secretion by naturally occurring inhibitors. The most significant of these are the tissue inhibitors of MMPs (TIMPs), which are locally produced by host tissues and even tumor cells (E. C. Kohn et al., 1995).
TIMPs form complexes with MMPs thereby preventing proenzyme activation and MMP catalytic activity.
Blockade of MMP production and/or activity has been shown in vitro and in vivo to prevent tumor growth and invasion (I. C. Anderson et al, 1996; X. Wang et al, 1994; A. Albini et al, 1995; A. Melchiori et al, 1992; A. Albini et al, 1994; R. Benelli et al, 1994), angiogenesis (R. E. Galardy et al, 1994; M. A. Moses, 1993), and cartilage/bone destruction in arthritis (J. G. Conway et al, 1995; E. M. O""Byrne et al, 1995; L. J. Bonassar et al, 1995).
Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by the placenta and other normal and neoplastic tissues. It is secreted and acts so in both an endocrine and paracrine way. hCG is an heterodimer composed of noncovalently bound subunits xcex1 and xcex2, each one having structural homology with the cystine-knot family of growth factors (platelet derived growth factor PDGF, vascular endothelial growth factor VEGF, nerve growth factor NGF and others), which are unrelated with hCG or with the family of glycoprotein hormones, as it has been recently demonstrated by the analysis of the hCG crystalline structure.
Dissociated hCG subunits can be found in urine and are often produced by tumors of the bladder, pancreas, cervix, lung, liver, and stomach as well as by trophoblastic tumors and testicular germ cell tumors.
Besides dissociated subunits, a variety of other metabolic forms of hCG can be found, particularly in urine, including hCG isoforms with various degrees of glycosylation, nicked hCG and a typical hCG-xcex2 proteolytic fragment, termed xcex2-core which is a disulfide bound two-chain polypeptide encompassing residues 6-40 and 55-92 of the hCG-xcex2 subunit.
In addition to the above mentioned isoforms, deglycosylated hCG can be obtained by different chemical and/or biotechnological ways, as described, for example, by Kalyan and Bahl (see Kalyan et al., 1983).
xcex2-core, as deglycosylated hCG or the single isolated subunits, does not show hCG-like biological activity, but represents a major form of immunoreactive hCG and can contaminate pharmaceutical preparations of urinary hCG.
Lunardi-Iskandar et al. (Lunardi-Iskandar et al., 1995) have shown that hCG and the hCG xcex2-subunit inhibit KS cell lines derived from Kaposi""s sarcoma, and inhibit tumor production by such cell lines in nude mice. Regression of Kaposi""s sarcoma has been shown in two women during pregnancy, when the level of this hormone is high.
Applicant had also previously found (see Italian Patent Application No. RM96A309) that KS cells membranes above defined contain binding sites of xcex2-core and deglycosylated hCG and it has been concluded that xcex2-core itself and/or deglycosylated hCG is the agent responsible for the in vivo and in vitro biological activities found by Lunardi-Iskandar.
Object of the present invention is the use of hCG, hCG xcex2-subunit, as well as active fragments and derivatives thereof, or hCG xcex2-core in the preparation of a medicament for the prophylaxis and/or the treatment of pathologies requiring inhibition of the MMP enzymatic activity. Examples of such pathologies include: tumors, chronic inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, periodontitis, multiple sclerosis and other angiogenesis-related pathologies.
Another object of the present invention are pharmaceutical compositions comprising hCG, hCG xcex2-subunit, as well as fragments and active derivatives thereof, or hCG xcex2-core in the presence of one or more pharmaceutically acceptable excipients, for the prophylaxis and/or treatment of pathologies requiring inhibition of the MMP enzymatic activity.
A further object of the present invention are methods for the prophylaxis and/or the treatment of pathologies requiring inhibition of the collagenase enzymatic activity comprising administering an effective amount of hCG, hCG xcex2-subunit, as well as fragments and active derivatives thereof, or hCG xcex2-core together with a pharmaceutically acceptable excipient.
For human therapy, the preferred dose of active ingredient is comprised between 10,000 and 500,000 Units.
The administration route of this active ingredient can be intravenous, intramuscular and subcutaneous. Other administration routes, which can guarantee the desired blood levels of active ingredient, are equally comprised in the present invention.
HCG is, preferably, recombinant and can be prepared, for example, by expression from CHO (Chinese Hamster Ovary) cells, that have been suitably transfected with the corresponding DNA, according to the technique described in EP Patent 160,699.
Suitable lyophilized pharmaceutical compositions containing hCG are described in the published PCT Patent Application WO 93/11788 and can be advantageously used according to the present invention.
Liquid pharmaceutical formulations, ready to be used, are described in the PCT Patent Application No. 95/EP/1778 and can also be advantageously used according to the present invention.
The present invention will now be described by means of the following Examples which should not be construed in any case as limiting the invention. The Example will refer to the Figures, as specified.